Method for treatment of toxo-plasmosis in animals



United States Patent 3,275,508 METHOD FOR TREATMENT or ToXo- PLASMOSIS IN ANIMALS Kiyoshi Tsunoda, Kodaira-shi, and Noriyuki Okuda,

Tokyo-to, Japan, assignors to Daiichi Seiyaku Kabushiki Kaisha, Tokyo-to, Japan, a joint-stock company of Japan No Drawing. Filed July 6, 1964, Ser. No. 380,634 Claims priority, application Japan, July 16, 1963, 38/38,578

8 Claims. (Cl. 167-53) This invention relates to armethod for the treatment of toxoplasmosis in animals with 4-methoxy-6-sulfanilamidopyrimidine or its derivatives represented by the following formula:

CHQO

of the host in approximately ten days, while chronic infection is highly contagious, and causes great" misery to the infected. Therefore, with the progress of fundamental study being achieved'on animal toxoplasmosis, increasing public attention has been paid to this parasitic infection.

Although a few medicines have so far beentried for the treatment of animal toxoplasmosis, none has ever proved satisfactory.

The present invention has for its object the provision of a method for the treatment of toxoplasmosis in domestic or farm animals such as cattle, dogs, cats, swine, cows, sheep, horses, and birds.

We have conducted tests of numerous chemicals for efficacy with respect to animal toxoplasmosis and, as a result, have found that 4-methoxy-6-sulfanilamidopyrimidine and its derivatives can exe 'extraordinan'ly good therapeutic effect as compared with other ordinary medlcmes.

The compound, 4-methoxy-6-sulfanilamidopyrimidine which is used in the present invention has been synthesized for the first time by one of us, and for, which a patent application has already been filed (US. Patent Application Serial No. 132,591 (1961)). 4-methoxy-6 sulfanilamidopyrimidine recrystallized from methanol is in the form of colorless needless of M.P. 200-201 C. (uncorrected). An alkali salt of this compound is obtained by neutralizing 4-methoxy-6-sulfanilamidopyrimidine with caustic alkali, while an alkaline earth metal salt of this compound is readily obtained by treating the alkali metal salt thereof with an equivalent amount of alkaline earth metal chloride. 6-(N -acetylsulfam'lamido)-4- methoxypyrimidine which is also used in the present invention is produced by acetylating 6-(p-nitrobenzenesulfonamido)-4-methoxypyrimidine or its alkali metal salt to produce 6-(N -acetyl-p-nitrobenzenesulfanilamido)-4- methoxypyrimidine, and then by reducing the latter.

Patented Sept. 27, 1966 The outstanding effects of 4-methoxy-6-sulfanilamidopyrimidine and its derivatives upon toxoplasmosis were confirmed by tests conducted on mice. Unlike other animals, the mouse is said to be slow in the production of antibodies following infection with toxoplasma, the production reaching the maximum (according to Sabin and Feldmans dye test) after more than 30 days subsequent to the infection. Thus, mice once infected with a toxic strain such as RH strain will present symptoms of toxoplasmosis and die without exception Within a certain period of days. Therefore, the efficacy of the compounds of the present invention can be determined very conveniently by observing how long the survival time of mice can be prolonged by administration of said chemicals. It may be pointed out in this connection that, if a mouse dosed with a certain chemical for a period of from 6 to 14 days can survive for two weeks, the chemical is generally deemed effective, while, if it can survive for three weeks with the same total dosage, the chemical is regarded as being highly efficacious. From experiences with other drugs, it is apparent to us that the chemicals efiicacious for mice are all the more valuable to other animals which can more readily produce antibodies.

A large number of chemicals tested heretofore are ineffective, and even if they are effective and permit a limited number of mice to survive for a long period of time, a small'numb'er'of the protozoa remain alive in the bodies of the mice, often in the form of cysts. On the other hand, the infected mice administered with 4-methoxy-6-sulfanilamidopyrimidine survived for 51 days and were considered to be protozoa-free because, when the homogenized brains of these mice were subjected to blind passage tests over two generations, the inoculated mice showed no symptoms of toxoplasmosis.

Recently, serious consideration has been given to the possibility of placental infection of toxoplasmosis, but there is no record of treatment with ordinary drugs for this type of infection. Experimental treatment of preg nant mice with the compounds of the present invention, however, gave the same results as those in treatment of non-pregnant mice. All of the mice which survived had normal parturitions and nursed normal litters of young. These young were observed for 60 days, and none was found to be infected. The unmedicated controls were invariably infected ou the 5th day, and died on the 6th to 7th day.

For the test, a toxic strain separated from ascites of infected mice was used, and a suspension in saline was prepared in such a manner that a certain number of toxoplasma organisms were contained therein. A certain quantity (8.7Xl0 -l.0 10 of the suspension was injected .into the abdominal cavities of the test mice. The mice tested, 'both male and female, weighed from 17 to 19 g. Most of the pregnant mice used were from 20 to 22 g. in weight. The compounds were administered for seven consecutive days after the date of the infection. The compounds were mixed in with the diet, in five different percentages of 0.24, 0.12, 0.06, 0.03, and 0.015. As the diet, the formula feed for the mice was given in an amount of 4 g. per mouse per day. Drinking water was The elficacy of the drugs according to the present invenl l Toxoplasmosis in swine 3 exact efficacy because of due to the infection and also because the cysts with 4-methoxy-6-su1fanilamidopyrimidine 5 according to the following procedure.

tion was determined on the basis of the difference between the medicated mice and controls in the fatality and prolongation of the survival time which were observed daily throughout the whole testing period. The surviving mice were killed on the 51st day after the infection,

{and each 0.4 cc. portion of their homogenized brain was mice so injected were further observed for 30 days. If the mice were not infected during this period, they were killed, and again their homogenized brain was inoculated into another group of six mice (i.e. second generation), }and observation was continued for another period of 30 ldays. In the case when the mice were not infected during the period, those which survived after the first medijcation were considered to have been completely cured, with extinction of toxoplasma. None of the .test mice dosed with the drugs of the present invention was infected. .FOI' experiments with pregnant mice, those whose abdomens were evidently swollen, although the dates of fertilization were not known, were collected. In the gexperiments, the young born by normal parturitions were lobserved for not they were congenitally infected. All exhibited negative results.

the high fatality of swine in the infected meat pose a public health problem.

Experimental treatment of toxoplasma-infected swine was conducted injected into the abdominal cavities of six mice, and the must be cured with especially 60 days after birth, to observe whether or i caustic soda, the pH value of the resulting solution being adjusted to 9.041. Practically, a' daily dose of from l mg. to 100 mg./kg. is given by injection or in mixture with feedstutfs. In the latter case, the compound is pound and are non-toxic to, the animals, e.g. sodium bicarbonate, lactose, dextrose, sucrose, starch, talc,.calcium carbide, and shell powder, for the sake, of convenience in administration or to increase stability of the chemical, or the compound is directly mixed with feedstuffs such as wheat 'bran, rice bran, or soybean cake.

The drug may also housed in the form of a syrup, in whichcase less bitter 4-(N eacetylsulfanilamide)-6-methoxypyrirnidine is used as the principal constituent in admixture with a non-toxic pharmaceutical carrier.:

In order to indicate still more fully the nature of the present invention, the invention is described hereinbelow with respect to the following examples which are intended to be illustrative but in no way restrictive.

Example 1 Groups each of five mice weighing from 17 to .19 g. each were infected with 8.7 X 10 toxoplasma (RH strain) per mouse.

mixture with feed in such were as is shown in Table 1.

TABLE 1.;EXPERIMENTAL TREATMENT OF MICE Days after infection Dosage No. of mice Survival Group No. (Percent) 6 7 15 19 21 24 25 27 survived 50 rate days (Percent) No. of dead mice in each group Test swine about 40 days after birth were infected with toxoplasma which was isolated from naturally-infected swine. The swine were dosed with 4-methoxy-6-sulfanilamidopyrimidine by intramuscular injection immediately. after the infection and after development of the. symptoms. mals were in a state of heavy infection with an increasedtiter, but it was ascertained, nevertheless, that toxoplasma and its cysts had disappeared in each animal. This in-. dicates that, whereas the chemicals tried heretofore merely compound of the present invention has a thorough remedial eifect as it not only cures animal toxoplasmosis but causes complete extinction of toxoplasma. One of unmedicated controls died in 10 days after it exhibited symptoms of the infection, while another control showed severe symptoms and thereafter recovered. In either case, however, the existence of toxoplasma was confirmed in almost all internal organs of these test animalsi In the treatment according to the present invention, 4- methoxy-o-sulfanilamidopyrimidine may be administered to animals infected with toxoplasma,. either orally or otherwise. For example, the compo'und'may be given by intravenous, intramuscular, or hypodermic injection, or by mixing it into feedstuffs. For emaciated animals having little appetite, tion, the compound is diluted with a example, a sterilized solvent such as water. Preferably the compoundis dissolved in an aqueous solution of According to a subsequent dye test, the test ani- 7 alleviate the condition of toxoplasmosis in animals, the

injection is preferred. For injecsterilized liquid, for

As will be seen from the above table,fa-ll of the mice of the infected butunmedicated group No. 6 presented symtoms of toxoplasmosis in 5 days after the infection, and died on the 6th or 7th d-ay. mice, numerous toxoplasma organisms were observed:

The group No. 5 given a dosage of 0.015% symptoms of or 15th day. 0.06% group (No. 3),.0.12% (No. 1) group all survived for 18 days, deaths on the 19th day and thereafter. were .due to recurrence of the disease following discontinuation of medication after the first week. The number of dead mice was recorded during the, 19th to 27th day -after the infection. survived the fatal period were all sound and vigorous, and toxoplasma in the ascites of these animals were far less in number than those. in the control group.:

The test mice-of the 0.03% group (No. 4),

with sporadic The mice which survived for 50 days were killed and and homogenized brain was,

dissected on the 51th day,

obtained from each group. Then, groups of 6 mice were 'brain per mouse and were plasma.

groups observed. Next, the mice subjected to the above last experiment were killed and dissected, and homoge: nized brain was prepared in eachgroup. Groups of 6 mice were inoculated abdominally with 0.4 cc. of I the and observed for 30 days homogenized brain per mouse,

For 7 days from the date of-the infection, V inclusive, 4emethoxy-6-sulfanilamidopyrimidine was given to the test animals in thorough amounts that the percentages of the compound in the. diet were 0.24, 0.l2,'0.06,-0.03, and 0.015. The results i In the ascites ofthe dead presented 1 toxoplasmosis, and all .died on the 14th group (No. 2), and 0.24% I These deaths period from the. I The. mice which.

6 Example 3 3 groups of 2 swine about 40 days after birth were infected, by abdominal inoculation, with 20.5 X toxoplasma (HG strain) per swine. 'I hen, 4-methoxy-6-sul- TABLE 2.EXA M1NATION FOR TOXOPLASMA IN MICE INOCULATED WITH HOMO GE- NIZED BRAIN OF MICE WHICH SURVIV ED UPON TREATMENT WITH THE COMPOUND OF THE PRESENT INVENTION Dosage Mice No. of mice inocu- No. of mice which No. of mice inocu- No. of mice which (Percentage survived lated with homogpresented symlated again with presented symin feed) enized brain toms days later homogenized brain toms 30 days later No. 1 ((0.24%)- 2 6 O 6 0 N0. 2 (0.12%)" 2 6 0 6 0 No. 3 (0.06%) 4 6 0 6 0 N o. 4 (0.03%)" 2 6 0 6 0 From the above results, it is considered that, in this example, toxoplasma in the mice which survived after infection for more than 27 days had become extinct.

fanilamidopyrimidine was administered to the infected animals. The method, dosage, and duration of the medication were as is shown in Table 4.

TABLE 4.METHOD, DOSAGE, AND DURATION OF MEDICATION Example 2 Of the mice raised, those which were apparently pregnant, were collected, and were inoculated with 1.0 10 toxoplasma per mice. Then, groups of 5 mice were medicated for 7 consecutive days from the day of infection, inclusive, with 4 methoxy 6-sulfanil-aimdopyrimidine thoroughly mixed in feed in such amounts that the percentages of the compound in the feed for five diiferent groups were, respectively, 0.24% (for the group No. 1), 0.12% (No. 2), 0.06% (No. 3), 0.03% (No. 4), and 0.% (No. '5). The results were as isshown in Table 3.

The effects were estimated by the degrees and extents of clinical symptoms, conditions of recovery, results of autopsy, and extinction of toxoplasma.

All of the swine inoculated with toxoplasma were attacked by fever of from 40 to 41 C. on the 3rd day of the infection. Then medication for the swine No. 3 and No. 4 was begun on the 4th dayof the infection. The two test animals recovered on the 5th day of the medication. The swine No. 5 and No. 6 which were medicated immediately upon infection showed no symptoms of the disease. Of the test swine, one dead swine was dissected TABLE 3.EXPE RIMENTAL TREATMENT OF PREGNANT MICE Days after infection Group Dosage No. of mice Survival Rate of 0. (percent) 5 6 7 18 21 24 28 survived rate normal days (percent) parturition N o. of dead mice in each group When the mice delivered of their young died of toxoplasmosis, the young also died because of their inability to suckle. However, the young of the mother mice which survived grew normally. .Observation was continued for 60 days after birth of :14 young mice, that is, 8 and 6 born, respectively, 'by the two surviving mice of the group No. 1; of 14 young mice, that is, 4, 5, and 5 born by the 3 surviving mice of the group No. 2; of 25 young mice, that is, 3, 6, 6, and 10 mice born by the 4 surviving mice of the group No. 3; and of '12 young mice, that is, 4 and 8 born by the 2 surviving mice of the group No. 4. All grew soundly with no symptom of toxoplasmosis.

immediately after death, and the 7 surviving swine were killed and dissected on the 74 th day after the infection. The internal organs of all of the test swine were homogenized. The homogenized organs were subjected to blind 7 found in the bodies of the plasma and treated with the chemical.

TABLE 5.EXISTENCE OF TOXOPLASMA ORGANISMS. IN THE INTERNAL swine inoculated with tom- ORGANS OF INFECTED SWINE CONFIRMED BY :INOCULATlON INTO MICE Note: +=Toxoplasma demonstrated directly by mice.

=l==Toxoplasma-positive mice/inoculated mice. Toxoplasma-negative.

- Meanwhile, in the unmedicated control group, the swine No. 2 died on the 13th day, and existence of toxoplasma was confirmed in all of the internal organs examined, while the swine No. 3 survived the infection and substantially regained its health !but demonstrated existence of numerous toxoplasma in'its'internal organs excepting the liver, the lungs, and the pancreas.

We claim: I

1. A method for the treatment of toxoplasmosis in animals which comprises dosing an infected animal. with at least one of the compounds of the formula:

vherein R is a member selected from the group consisting )f hydrogen, alkali metal, and alkaline earth metal.

2.. A method for the treatment of toxoplasmosis in inima-ls which comprises dosing an infected animal with.

he compound 4-methoxy-6-sulfanilamidopyrimidine of he formula:

N NS OPQ-NH: E

CH O

herein R is a member selected from the group consisting hydrogen, alkali metal, and alkaline earth metal, said )mpound being dissolved in a sterilized diluent.

Internal Organs oi Swine Seroreaction Pig N o. Dye test Dye test Brain Diaphragm Liver Kidney Spleen Lungs Pancreas Lymph- Myocar- Muscles titer of titer of glandula dium Swine inoculated mice 4 a x 4096 x 4 component.

A: Not determined, because of toxoplasma demonstratiom )=D1rect examination of cysts in mouse brain.

mg.-/kg. (dosage weight per kg. of animal ,bodywherein R is a member selected from the group consisting of hydrogen, alkali metal, andalkaline earth metal, said 1 compound being dissolvedin an. aqueous. solution'of caustic soda, the pH of which solution is adjustedto 9.0-1 1.0, and being administered in a dosage of from 20 mg/kg. to 100 m-gJkg. (dosage weight per kg. of animal body weight) a day in terms of the principal cornponent.

7. A method according to claim 4, wherein the injection:

is given. intramuscularly.

8. A method for the treatment of toxoplasmosis in animals which comprises administering to an infected animal a syrup made of 4-(N -acetylsulfanilamido)-6-methoxy-" pyrimidine of theformula:

' omo OCH and a non-toxic pharmaceutical carrier, in a dosage of from 20 nag/kg. to 100 rag/kg. (dosageweight per kg. of animal body weight) a day in terms of the principal References Cited by the Examiner FOREIGN. PATENTS 9/1960 Belgium. '9/1960 Belgium.

NORRIS G. MANN, STANLEY I. FRIEDMAN;

- Assistant Examiners. 

1. A METHOD FOR THE TREATMENT OF TOXOPLASMOSIS IN ANIMALS WHICH COMPRISES DOSING AN INFECTED ANIMAL WITH AT LEAST ONE OF THE COMPOUNDS OF THE FORMULA: 